Change In High Mobility Group Box-1 Protein Expression And Its Role In Mediated Multiple Organs Dysfunction After Severe Injury.

Objective: High mobility group box 1 protein (HMGB1) as a late-acting cytokine mediates lethality of sepsis and systemic inflammation. The present study was performed to determine the expression patterns of HMGB1 and to investigate the influence of EP and Xuebijing on HMGB1 expression on multiple organs dysfunction in rats with delayed resuscitation after severe thermal injury, and to clarify the potential mechanism of postburn immunosuppression related to HMGB1 induction, in turns preventing the development of sepsis and subsequent multiple organ dysfunction syndrome following severe injury.Methods: A widely used technique for induction of full-thickness scald burns was used in this study. Rats were anesthetized, and the dorsal and lateral surfaces of the rats were shaved. Rats were placed on their backs and secured in a protective template with an opening corresponding to 30% of the total body surface area, and the exposed skin was immersed in 99°C water for 12 s. Sham-injured rats were subjected to all of the procedures except immersion in boiling water.Male Wistar rats were subjected to 30% full-thickness scald injury and delayed resuscitation after 6 hours. One hundred and fourteen rats were randomly divided into five groups as follows:①normal control group (n=6): animals were killed after anesthesia;②sham group (n=18): animals were killed on hours 8, 24, and 72 .animals were treated with normal saline(4ml) intraperitoneally at 12, 24, 36, 48 and 60 hours after sham burn (37C°);③b urn group (n=30), animals were treated with Ringer's solution (3ml) intraperitoneally at 2,12, 24, 36, 48 and 60 hours after burn injury;④E P (Ethryl pyruate) treatment group (n=30), animals were treated with the Ethyl pyruate intraperitoneally at 2 ,12, 24, 36, 48 and 60 hours after scald injury.⑤Xuebijing treatment group (n=30), animals were treated with the Xuebijing(4ml/kg) infused with vein of penis at 2 ,12, 24, 36, 48 and 60 hours after scald injury. The last four groups were administrated with 40ml/kg of normal saline intraperitoneally at 6 hours after burn, and divided into three subgroup, sacrificed on the 8,24,72 hours after scald injury. Animals were sacrificed at designated time points, and blood and tissue samples from livers, lungs, kidneys as well as small intestine were harvested aseptically to determine organ damage related variables and levels of various cytokines.In addition, additional experiments were were performed to study the influence of EP and Xuebijing on survival time of rats after severe thermal injury,The mortality of rats in each group was recorded up to 7 days.HMGB1 and TNF-αmRNAs expression in tissue samples from livers, lungs, kidneys were semi-quantitated by the reverse transcription polymerase chain reaction (RT-PCR) taking GAPDH as an internal standard. Protein expression of HMGB1 was detected by Western blot and immunohistochemistry. TNF-αLevels in tissue samples from livers, lungs, kidneys were determined by commercial available enzyme-linked immunosorbent assay (ELISA) kits for rats. The major organ functional indices, the hepatic functional indices—serum AST and ALT contents and the renal tissues functional indices—serum BUN and Cr contents were measured with automatic biochemistry analyzer, and lung tissue myeloperoxidase (MPO) and small intestine diamine oxidase (DAO) were also measured. Tissues pathological changes of the livers, lungs, kidneys were examined using HE staining.Results: 1. HMGB1 mRNA levels significantly increased in various tissues during 8-72h after severe thermal injury with delayed resuscitation, peaking at 24h, kept high values up to 72h post-injury. HMGB1 protein expression markedly enhanced in liver tissues during 8-72h and in lung tissues at 8-72h following scald. Kidney tissues HMGB1 protein expression showing elevated at 24h post-injury. HMGB1 levels were significantly decreased at corresponding hours in tissues by EP treatment group compared to Scald controls. HMGB1 levels were significantly decreased at corresponding hours in tissues by Xuebijing treatment group compared to scald controls.2. TNF-αmRNA and protein expression was significantly augmented in the liver and lung tissues at 8h following scald, and recovered at 24h. TNF-αmRNA and protein levels in the liver and lung tissues significantly increased with two peaks at 8h and 72h.Meanwhile, kidneys TNF-αmRNA and protein markedly increased at 8h following Scald and kept high values up to 24h. Treatment with EP could significantly reduced TNF-αmRNA and protein levels in liver, lungs and kidneys at compared to Scald controls. Moreover, treatment with Xuebijing could decrease TNF-αmRNA and protein levels in liver and lungs, without marked influence on hepatic TNF-αmRNA and pulmonary TNF-αprotein levels at 8h after injury. Treatment with Xuebijing also could significantly inhibit kidney TNF-αmRNA and protein expression at 24h post-injury.3. Compared with sham scald controls, levels of serum ALT, AST, Cr and BUN and pulmonary MPO activities were significantly increased with certain extent at different intervals in rats with delayed resuscitation after scald injury. Both levels of AST,ALT and Cr of serum and pulmonary MPO activities were markedly enhanced following scald, peaking at 24h post-injury. Levels of serum AST and Cr showing persistent elevated up to 72h .On the contrary, intestinal DAO activities were significantly decreased at 8-24h after scald. Treatment with EP could markedly down-regulate levels of serum AST and ALT at 8, 24 and 72h following scald. Meanwhile levels of Cr of serum at 24h after injury and BUN of serum were markedly decreased at 8h and 24h after scald. Moreover, using Xuebijing treatment could markedly reduce levels of serum AST and ALT at 8, 24 and 72h after scald, and decrease level of serum Cr at 24h, 72h and level of serum BUN at 24h following scald, except AST level at 8h post-injury. Administration of EP could markedly decrease pulmonary MPO activities, and increase intestinal DAO activities at 8h and 24h following scald .Treatment with Xuebijing could markedly down-regulate pulmonary MPO activities at 24h, and up-regulate intestinal DAO activities at 8h-24h following scald .4. Many inflammatory cells in tissues were observed using light microscope at 8, 24 and 72h following scald. The histological morphology of tissues injury was ameliorated after administration of EP and Xuebijing compared with sham scald controls. EP and Xuebijing protect tissues against acute injury induced by severe thermal injury. 5. Furthermore, treatment with EP and Xuebijing could significantly improve the 1- to 7-day survival rates in animals subjected to severe thermal injury.Conclusion: 1. Severe thermal injury with delayed resuscitation could increase tissues HMGB1 expression and release in rats. HMGB1 is produced lately and decreases slowly, which is identified as a"late"inflammatory mediator. HMGB1 might play an important role in development of excessive inflammatory response and subsequent multiple organ damage.2. At the early stage of scald injury, scald-induced TNF-αrelease could stimulate and up-regulate HMGB1 expression, TNF-αexpression might be involved in synthesis and release of HMGB1 associated with excessive inflammatory response challenge in local tissues. On the other hand, HMGB1 might also influence TNF-αformation during late phase in rats with delayed resuscitation after scald injury, implying the interaction between HMGB1 and TNF-αduring systemic inflammation.3. Treatment with EP and Xuebijing could inhibit the excessive expression of HMGB1 and TNF-α, thereby preventing the development of uncontrolled inflammatory response and progressive multi-organ dysfunction following scald. It also prolong survival time of rats with severe thermal injury. EP and Xuebijing have a therapeutic potential for suppressing inflammatory response disorder induced by HMGB1 and improving multiple organ injury.